1. Technical Field
The present invention relates to automated focusing systems, in particular to extremely rapid automatic focusing of optical scanning systems.
2. Description of the Related Art
With the development of combinatorial chemistry and bioassays, automated imaging is becoming extremely important. A large variety of tests can be conducted with such systems, such as are disclosed in WO 99/08233 and WO 98/47006. Some of these tests, particularly ones based on fluorescence and reflected light, use confocal systems such as those disclosed in U.S. Pat. No. 5,900,949. In such a system, light is applied through the optics of the system to excite a sample to fluoresce or phosphoresce, or simply to reflect the light. The resulting emitted, reflected or scattered light then is detected either through a separate optical system to the side of the light source, as shown in U.S. Pat. No. 5,900,949, or through reflection or emission back through the same initial optical system as the light source, by way of a half-silvered mirror or di-chroic beam splitter.
In a typical scanning system (illustrated in FIG. 1) a focused beam of light moves across a sample and the resultant reflected or fluorescent light is detected. A fluorescent system typically includes a source of light 10 at the proper wavelength, xcexex, to excite the sample or a dye in the sample. This light is focused through source optics 12 and deflected by mirror 14 via scan lens 26 onto sample 16. Light that fluoresces or is reflected from the sample returns to detection optics 18 via half silvered mirror or di-chroic beam splitter 15. Alternatively, the emitted or fluoresced light can be detected from the side of the system, as shown in U.S. Pat. No. 5,900,949. Light passing through detection optics 18 then is detected using a CCD or equivalent element 20, the output from which is provided to computer 22 for analysis. Motor 24 is used to move mirror 14 to scan the excitation beam across the sample 16. The excitation beam, motor, optics and the rest of the system then are controlled by computer 22 to scan relevant portions of sample 16.
In a true confocal system, the system will reject light that is not substantially in focus. As illustrated in FIG. 2, the light in such a confocal system typically will be deflected by mirror 14 through scan lens 26. A confocal system typically has a very small depth of field d, as illustrated in FIG. 2. Sample 16 is in scan field 29, that is, in the depth of field d, for a scan across sample 16, traversing the range of scan. The focal length of the system is f, and the relative sizes of the values are f greater than  greater than d greater than  greater than xcex. The range of scan may vary from tens of micrometers to centimeters, depending on the system.
For a truly flat and level surface in a confocal system, once the collection system and the sample are brought into focus, no more focusing along the +z or xe2x88x92z axis (up or down, as shown in FIG. 2) is required. If the light beam is scanned, the assumption is that the design of the system is such that rotation of mirror 14 does not move the light beam out of the nominal plane of focus, i.e., scan field 29 is essentially flat in the area where the sample is located.
As will be apparent, the sample must be kept continuously in focus during a scan. One technique for doing this automatically or manually brings the sample into focus below a stationary focused beam, only once, and then scans the sample by moving it on an x-y translation stage. The distance from the sample to the objective then remains constant since the sample does not move up or down, throughout the scan. This method is used by several imaging manufacturers.
Autofocus systems for optical scanners often use a half-blocked or obscuration technique to bring the sample into focus, such as is shown in FIGS. 3a, b, c, and described in detail in section 31.4 of the Optical Society of America""s Handbook of Optics Vol. 1 (on CD-ROM), published by McGraw Hill (1996). In such a system, light 41 reflected or emitted from sample 16 passes through a lens 26. Most of the light passing through lens 26 then will be directed to the detection optics for analysis, but, as shown in FIG. 1, some of the light will be directed by a low reflection beam splitter 40 to the autofocusing system 42, shown in detail in the upper portions of FIGS. 3a, b, c. (For clarity of illustration, beam splitter 40 is omitted from FIGS. 3a, b, c.)
If sample 16 is in focus, as shown in FIG. 3b, light 41 is collimated, so that light 47 passing through lens 46 is properly focused on focal point 49. If sample 16 is too far from lens 26 (the xe2x88x92z direction), light 41 tends to converge too much, as shown in FIG. 3a. If sample 16 is too close to lens 26 (the +z direction), light 41 tends to diverge too much, as shown in FIG. 3c. 
In a typical autofocusing system, half of the beam of light 41 is blocked by knife edge 44. The remaining portion of light 41 passes through lens 46 to become light 47 and impinges upon photodetector 48. Photodetector 48 typically has halves A, B centered on the focal point 49 of the photodetector 48, with each half A, B serving as an independent detection region.
When properly focused, as shown in FIG. 3b, the light 47 impinges upon the center 49 of the photodetector 48, between halves A, B, or at the very least impinges equally upon halves A, B. In contrast, when sample 16 is too far from lens 26, as shown in FIG. 3a, more of light 47 impinges on photodetector portion B than on A, and similarly, as shown in FIG. 3c, when sample 16 is too close to lens 26, more of light 47 impinges on photodetector A than on B. Therefore, the position of sample 16 relative to lens 26 can be determined by analyzing the relative signal strengths being generated by photodetector portions A and B. This can be done through any suitable method, but is conveniently done by subtracting the values of the outputs of the two portions A and B of the photodetector in circuit 51 to generate a Focus Error Signal (FES) 50.
In theory, the absolute value of FES 50 is indicative of the distance by which sample 16 is out of focus, while the positive or negative value of FES 50 indicates the direction in which the sample 16 is out of focus. When sample 16 is in focus, light 47 either impinges on photodetector center 49, or at least is equally balanced between portions A, B, with the result that the value of FES 50 is 0 and no z-axis adjustment is needed (it will be understood that the value need not be exactly 0xe2x80x94some range around 0 will normally be considered equivalent to 0). If more of light 47 impinges on half B of the detector (as shown in FIG. 3a), FES 50 is a positive signal, indicating that the z translation stage is off in the xe2x88x92z direction, so the stage should be moved in the +z direction to bring the system into focus. If sample 16 is too close to lens 46, more light 47 hits A than B, and FES 50 is negative, indicating that the stage is out of focus in the +z direction, and should be moved in the xe2x88x92z direction to bring the system into focus. Z-axis translation stages responsive to such an FES signal in this fashion are commercially available.
While working with new materials to hold samples, the inventors encountered problems with focusing the prior art systems when the sample surface itself undulates significantly. As illustrated in FIG. 4, if the surface of sample 16 is not smooth, portions of the surface may be out of focus, even though sample 16 as a whole stays at the same distance from the lens 26. This may be true of both scanning beam and scanning stage systems.
Specifically, FIG. 4 illustrates a set of DNA oligonucleotide probes 30 deposited on the surface of substrate 16. Such probes often are chemical systems which combine with immobilized clipped DNA fragments to identify the presence or absence of various DNA structures.
As will be apparent in FIG. 4, there is a base height h that is the minimum thickness of the substrate, but there is also a variability xcex94h in the height of the surface of the substrate. Depending on the substrate used, xcex94h can be considerably larger than the depth of field d for the focused light beam 34. As a result, DNA probes 30 may be in or out of focus even without any vertical movement of substrate 16.
Such substrate undulation can be minimized, but usually requires significant machining or use of fairly expensive materials such as silicon wafers or glass. Inexpensive materials, such those taught in WO 99/53319, are particularly likely to have such undulating surfaces, but are highly desirable for use.
Therefore, if as shown in FIG. 4 the sample surface height variation xcex94h is greater than the depth of field d, some system must be in place to keep probes 30 within depth of field d or regions of the image will be blurry in a non-confocal system, or dark in a confocal system. Such a system must be capable of refocusing easily at different depths, but at the same time, the system must be extremely fast. This can be done by moving the focusing lens, but is more commonly done by moving the stage, which might be piezoelectric stage, a stage mounted to a solenoid or voice coil, or a translation stage. In any case, the position of the stage is responsive to the output of the computer.
The difficulty is the number of times the system must be refocused. For example, to capture a 512 pixelxc3x97512 pixel image (a frame) in five seconds, the autofocus change must take no more than 19 micro-seconds for autofocus from pixel to pixel (5 seconds divided by 512xc3x97512 pixels). This requires an extremely rapid autofocus system.
The inventors recognized that in actual practice, the absolute value of the FES indicates the distance by which the sample is out of focus only if the sample is not too far out of focus. If the sample is too far out of focus, the direction can still be determined from the positive or negative value of the FES, but the distance to the proper focal position cannot be determined accurately, because the FES becomes saturated, that is, the FES reaches a plateau. When the FES is saturated, the absolute value of the FES indicates only the saturation level and no longer indicates the distance to the correct focal point. The system or operator must then xe2x80x9cguessxe2x80x9d how much to move the sample to get it into focus, i.e., move it by some arbitrary amount. If the initial move is not far enough, another guess is necessary, while if it is too far, a move in the opposite direction may be needed. This repeated guessing severely limits the speed with which the system can refocus as it scans from one point to the next.
The present invention therefore provides consistently rapid focusing by combining a half-blocked autofocus system with a variation in the amount of light being applied to the sample.
A first embodiment of the present invention accomplishes rapid focusing by determining when the FES is ambiguous, and modifying the amount of light reaching the photodetector when it is ambiguous. Specifically, if the FES is ambiguous, the diameter or area of the beam of light is reduced by some predetermined amount. This will then unsaturate the FES and allow the system to move the lens or sample quickly to a position relatively near the optimum focus. The light beam diameter or area then can be increased back up, and final focusing done.
A second embodiment of the present invention accomplishes rapid focusing by providing an additional mechanism for determining the approximate distance to the correct focal point. This may be done by providing a photodetector which can directly detect the radial offset of the light beam striking the photodetector, so the distance can be determined without relying on the absolute value of the difference between the signals on opposite sides of the photodetector.
Still further, both embodiments may be combined to maximize the distance over which the system can quickly focus.
With any of the above embodiments, the result is a two step process that can focus quite rapidly, and considerably more rapidly on average than done with a conventional half-blocked system alone. Combined with a very rapid z stage control, such as a piezoelectric fast response system, and suitably rapid electronic control circuitry or software, the system can achieve the rapid response time desired for scanning systems.
As will be apparent, having such a rapid autofocus will allow use of less expensive substrates with undulating surfaces, while at the same time allowing rapid image acquisition of the resulting product.